The solution structure of Co-Bleomycin (CoBLM) A2 green (the hydroperoxide form oj CoBLM) complexed with self-complementary oligonucleotide d(CCAGQCCTGG) with a cleavage site at C6 has been determined by 2D NMR spectroscopic methods and molecular dynamics calculations. Intermolecular NOEe (60 between CoBLM A2 green and DNA) and intrarnolecular NOEs (61 within CoBLM A2 green) have defined the position and orientation of CoBLM A2 green with respect to its single binding site in the duplex. These studies have provided the first structural insight into the mode of bonding of the bithiazole tail of CoBLM A2 green to DNA, the basis for specificity of its cleavage ai pyrimidines (Py) in d(G-Py) sequences, and the orientation of its terminal oxygen of the hydroperoxide [unreadable]relative to the 4' carbon hydrogen bond being cleaved in the DNA. The 4-amino group and the N3 of the pyrimidine ring of CoBLM A2 green form specific hydrogen bonds with the N3 and the 2-amino group, respectively, of the G5 in the duplex and provide an unusual example of a minor groove base triple-like interaction. A basis for the preference for G over A, 5' to the Py cleavage site, is thus established. The most remarkable feature of this structure is the observation of the proton associated with the hydroperoxide of CoBLM A2 green and its observed intermolecular NOEs to the minor groove protons of C6 and C7 of the duplex. Thus, this structure provides a rare snapshot of an analog of a reactive intermediate poised to initiate the hydrogen atom abstraction event. The molecular modeling reveals thai the distal oxygen of the hydroperoxide is 2.5 A from the 4'-hydrogen of C6. A number of additional intramolecular hydrogen bonds between the hydroperoxide ligand and the peptide linker region are also proposed, which appear to play a key role in positioning the reactive intermediate near the hydrogen atom being abstracted. This structural model makes a number of predictions that can be tested experimentally.